Biosensor, method of forming and use

ABSTRACT

A sensor device ( 1-5 ) has a sensing surface on which, in use, first molecules ( 5 ) are immobilized. The first molecules ( 5 ) are capable of interaction with second molecules which may be present in a sample of fluid applied to the sensing surface, such interaction resulting in a measurable change of some physical property of the sensor device. The sensing surface is coated with a layer ( 4 ) of diamond-like carbon to protect and preserve the integrity of the sensing surface.

This invention relates to improvements in or relating to sensors, and inparticular to those sensors termed biosensors, ie devices for theanalysis and investigation of biological or biologically active speciessuch as antigens and antibodies, enzymes, substrates, proteins, haptens,whole cells and cellular fragments and nucleic acids.

Many devices for the automatic determination of biochemical analytes insolution have been proposed in recent years. Typically, such devices(biosensors) include a sensitised coating layer which is located in theevanescent region of a resonant field. Typically, the coating layercomprises a layer of biological molecules chemically linked to thesurface, either directly or via an intermediate linking molecule, orimmobilised within a matrix of, for instance, hydrogel molecules boundto the surface.

Detection of the molecule under investigation (“the analyte”) typicallyutilizes optical techniques such as, for example, surface plasmonresonance (SPR) or frustrated total reflection (FTR), and is based onchanges in the thickness and/or refractive index of the coating layerresulting from interaction of that layer with the analyte. This causes achange in the properties of the sensor, eg a change in the angularposition of the resonance. Other forms of biosensor include devices withsemiconducting surfaces, the electrical properties of the device beingmonitored and, notably, acoustic devices in which changes in surfacebulk loading are detected.

Since the measurements made using biosensors of the types described areessentially measurements of events or changes occurring at thesensitised surface of the device, it is critical to the accuracy andreliability of the measurements that the integrity of that surface ismaintained. In an SPR or FTR sensor, for example, the device monitorsthe resonating properties of a structure the natural frequency of whichis altered as changes take place at its surface. If the sample can alterthe bulk of the structure in a non-specific manner then the integrity ofthe measurement is destroyed. In practice, maintenance of surfaceintegrity may not be achieved and this gives rise to errors in theexperimental results and/or greatly limits the useful life of the sensordevices. For example, repeated application of reagents to the surface(as is inevitable in a series of measurements) may result in attritionof the surface, with a consequent unpredictable change in properties.The surface may be somewhat porous, with the result that reagents may beabsorbed, again changing the properties of the device. Chemical linkagesbetween the surface and the molecules immobilized on it may also becomebroken in the course of chemical treatment.

There has now been devised an improvement to sensors of the kindgenerally described above which overcomes or substantially mitigates thedisadvantages of the prior art.

According to the invention, a sensor device has a sensing surface onwhich, in use, first molecules are immobilized, the first moleculesbeing capable of interaction with second molecules which may be presentin a sample of fluid applied to the sensing surface, such interactionresulting in a measurable change of some physical property of the sensordevice, wherein the sensing surface is coated with a layer ofdiamond-like carbon.

The sensor device according to the invention is advantageous primarilyin that the layer of diamond-like carbon (DLC) protects and preservesthe integrity of the sensing surface. The device is impervious to thereagents and fluids with which it is, in use, contacted. Problems ofattrition of the surface and porosity are reduced, and linkages of thefirst molecules to the surface are more stable. Furthermore, andparticularly importantly, by appropriate control of the composition ofthe diamond-like carbon layer (as described below) a wide variety offunctionalities may be incorporated into it in a thickness-dependentmanner.

DLC is a dense, partially sp³ bonded form of amorphous carbon. Itsatomic structure consists of a network of sp³ and sp² sites, theconnectivity of the sp³ sites controlling the mechanical properties ofthe material. DLC is conventionally used as a hard coating material, ieto confer “diamond-like” properties such as mechanical hardness and lowfriction on substrate materials. Since the purpose of the DLC layer usedin the present invention is not primarily to confer a high degree ofhardness on the active surface of the sensor device, the layer may havea hardness which is considerably less than that achieved in conventionalapplications of DLC.

The DLC layer may be formed by plasma deposition or chemical vapourdeposition techniques. Typically, monomeric starting materials in thegas phase are introduced into a vacuum chamber containing a pair ofelectrodes. The device to be coated is supported in the chamber on oneof the electrodes and a radiofrequency or microwave discharge isapplied.

Generally, the starting material includes a hydrocarbon, most preferablymethane. However, in principle any suitable hydrocarbon may be used, egethylene, acetylene, ethane, or aromatic species such as toluene andstyrene. Mixtures of starting materials may be used to give desiredphysical properties.

It may also be desirable to incorporate other chemical functionality inthe DLC layer. For instance, by introducing CH₃NH₂ gas in the finalstages of the deposition, a DLC layer may be formed with a surface whichincludes amino groups. Such groups may be useful for the directimmobilization of biomolecules. Similarly, inclusion ofcarboxylate-containing species in the vapour may give rise to a surfacewith carboxylate functionality. The starting materials may also includesmall quantities of gases such as argon, neon, nitrogen, oxygen orhelium. Appropriate combinations of starting materials may also be usedto produce DLC layers having particularly hydrophobic or hydrophilicproperties.

Because the polymerisation reaction is essentially simple, a high degreeof control can be exercised over the chemical and physical nature of theDLC layer, enabling the properties of that layer to be easily tailoredto the particular application for which the sensor device is intended.One physical parameter which is important is the density of the DLClayer, which is determined largely by the proportion of sp³ to sp²hybridized carbon. For optical sensors, a dense DLC layer is desirableto minimise the thickness of the DLC layer necessary to provide thenecessary degree of protection without adversely affecting the opticalproperties of the sensor. The density (and thickness) of the DLC layermay be less important, or not at all important, for non-optical sensors.

The DLC layer should have a thickness which is sufficient to confer thedesired degree of protection on the sensing surface of the sensordevice. The thickness of the DLC layer can be controlled by appropriatechoice of the operating parameters of the deposition apparatus, notablythe period for which the deposition is carried out. In general, thethickness should be no greater than the minimum required, so as to avoidany possible deleterious effects of the DLC layer on the properties, egthe sensitivity, of the sensor device. Typically, the DLC layer willhave a thickness of less than 100 nm, more preferably less than 50 nm,and particularly less than 20 nm. A thickness of greater than 1 nm, andgenerally greater than 5 nm will normally be required. The thickness ismost preferably of the order of 10 nm.

The DLC layer may be applied directly to the surface of the sensordevice which it is desired to protect. However, since the DLC layer maynot adhere sufficiently well to the material of that surface, it may benecessary to apply first a thin layer of another material to which theDLC layer will adhere well.

The sensor device according to the invention may be of any type, eg anoptical sensor or any other formn of sensor in which changes at thesensing surface result in a measurable change of physical property. Onepreferred form of optical sensor is based on frustrated totalreflection. The principles of frustrated total reflection (FTR) are wellknown; the technique is described, for example, by Bosacchi and Oehrle[Applied Optics (1982), 21, 2167-2173]. An FTR device for use inimmunoassay is disclosed in European Patent Application No 0205236A andcomprises a cavity layer bounded on one side by the sample underinvestigation and on the other side by a spacer layer which in turn ismounted on a substrate. The substrate-spacer layer interface isirradiated with monochromatic radiation such that total reflectionoccurs, the associated evanescent field penetrating through the spacerlayer. If the thickness of the spacer layer is correct and the incidentparallel wave vector matches one of the resonant mode propagationconstants, the total reflection is frustrated and radiation is coupledinto the cavity layer. The cavity layer must be composed of materialwhich has a higher refractive index than the spacer layer and which istransparent at the wavelength of the incident radiation.

An FTR sensor will generally include an optical structure comprising

a) a cavity layer of transparent dielectric material of refractive indexn₃,

b) a dielectric substrate of refractive index n₁, and

c) interposed between the cavity layer and the substrate, a dielectricspacer layer of refractive index n₂.

In use, the interface between the substrate and the spacer layer isirradiated with light such that internal reflection occurs. Resonantpropagation of a guided mode in the cavity layer will occur, for a givenwavelength, at a particular angle of incidence of the excitingradiation.

The angular position of the resonant effect depends on variousparameters of the sensor device, such as the refractive indices andthicknesses of the various layers. It is a pre-requisite that therefractive index n₃ of the cavity layer and the refractive index n₁, ofthe substrate should both exceed the refractive index n₂ of the spacerlayer. Also, since at least one mode must exist in the cavity to achieveresonance, the cavity layer must exceed a certain minimum thickness.

The cavity layer is preferably a thin-film of dielectric material.Suitable materials for the cavity layer include silicon nitride, hafniumdioxide, zirconium dioxide, titanium dioxide, aluminum oxide andtantalum oxide.

The dielectric spacer layer must have a lower refractive index than boththe cavity layer and the substrate. The layer may, for example, comprisean evaporated or sputtered layer of magnesium fluoride. In this case aninfra-red light injection laser may be used as light source. The lightfrom such a source typically has a wavelength around 600-800 nm. Othersuitable materials include lithium fluoride and silicon dioxide.

The refractive index of the substrate (n₁) must be greater than that(n₂) of the spacer layer but the thickness of the substrate is generallynot critical.

By contrast, the thickness of the cavity layer must be so chosen thatresonance occurs within an appropriate range of coupling angles. Thespacer layer will typically have a thickness of the order of severalhundred nanometres, say from about 200 nm to 2000 nm, more preferably500 to 1500 nm, eg 1000 mn. The cavity layer typically has a thicknessof a few tens of nanometres, say 10 to 200 nm, more preferably 30 to 150nm, eg 100 nm.

It is particularly preferred that the cavity layer has a thickness of 30to 150 nm and comprises a material selected from silicon nitride,hafnium dioxide, zirconium dioxide, titanium dioxide, tantalum oxide andaluminum oxide, and the spacer layer has a thickness of 500 to 1500 nmand comprises a material selected from magnesium fluoride, lithiumfluoride and silicon dioxide, the choice of materials being such thatthe refractive index of the spacer layer is less than that of the cavitylayer.

Preferred materials for the cavity layer and the spacer layer aresilicon nitride and silicon dioxide respectively.

At resonance, the incident light is coupled into the cavity layer byFTR, propagates a certain distance along the cavity layer, and couplesback out (also by FTR). The propagation distance depends on the variousdevice parameters but is typically of the order of 1 or 2 mm.

At resonance the reflected light will undergo a phase change, and it isthis which may be detected. Alternatively, as described in InternationalPatent Application No WO 92/03720 the cavity layer and/or spacer layermay absorb at resonance, resulting in a reduction in the intensity ofthe reflected light.

The DLC layer is preferably formed on the surface of the cavity layerafter the spacer layer and cavity layer have been applied to thesubstrate. In addition to the DLC layer, the cavity layer or the cavitylayer and the spacer layer may be formed by plasma deposition orchemical vapour deposition techniques. The substrate may, for example,be placed in the deposition chamber and the spacer layer, cavity layerand DLC layer formed sequentially.

Another particular form of sensor which may be mentioned is the type ofoptical sensor disclosed in copending International Patent ApplicationWO 97/29362. Such a device comprises a substrate having a waveguideformed on at least part of the surface thereof, the waveguide having afirst major surface which constitutes an interface between the waveguideand the substrate and a second major surface upon which the firstmolecules are immobilized, at least a region of the first and/or secondmajor surface being formed with a periodic refractive index modulation.When such a device is modified in accordance with the present invention,the DLC layer is applied to the second major surface of the waveguide,ie between the waveguide and the first molecules.

With such a device, as described in WO 97/29362, high intensity ofreflected light may be observed. Such high reflection may be termed“anomalous” or “abnormal” reflection. Interaction of the molecularspecies immobilized on the waveguide surface with analyte molecules in asample which is contacted with the waveguide causes a local change inrefractive index in the vicinity of the waveguide surface. This in turnchanges the angle of incidence or wavelength at which the reflectionmaximum occurs, providing a sensitive indicator of the chemicalinteraction taking place at the surface.

The periodic refractive index modulation is preferably a surface reliefprofile or a grating formed in the surface of the substrate to which thewaveguide coating is applied and/or in the surface of the waveguide onwhich the first molecules are immobilized. The periodic refractive indexmodulation may be formed in one or both major surfaces of the waveguide.

The grating may have a variety of forms. For example, it may besinusoidal, rectangular, triangular (saw-tooth) or trapezoidal.

The substrate is conveniently a chip, eg of glass or silica, and, inuse, the superstrate is most commonly an aqueous sample. The waveguideis preferably of relatively high refractive index, e.g. a materialhaving a refractive index of, say, 1.80 to 2.50. Suitable materials forthe waveguide include hafnium dioxide, silicon nitride, tantalumpentoxide and titanium oxide.

The optimal physical dimensions of the sensor device, grating etc willdepend on the wavelength of the incident light. In the followingdescription, the values given for the waveguide thickness, grating depthand period, light beam diameter etc encompass those suitable forcommonly-used wavelengths, eg a wavelength of 633 nm.

Typically, the wa,.eguide may have a thickness of the order of 50 nm to300 nm, more preferably 100 nm to 200 nm. We particularly prefer thethickness of the waveguide to be in the range 140 nm to 180 nm.

The depth of the periodic refractive index modulations (e.g. thecorrugations in the surface of the substrate) is preferably less than 50nm, more preferably less than 25 nm, eg typically 2 nm to 20 nm or 5 nmto 10 nm. The period of the grating is typically 300 nm to 1000 nm, morepreferably 600 nm to 1000 nm.

For use in the analysis of biochemical species, the first moleculesimmobilized, in use, on the sensing surface of the sensor device (ie onthe DLC layer) will generally be biomolecules, eg specific bindingpartners for the second molecules (the analyte). The first molecules maybe bound to the surface by methods which are well known to those skilledin the art. The first molecules may be covalently bound to the DLClayer, either directly or via linking molecules, or may be bound to amatrix, eg a porous matrix of a hydrogel such as agarose or dextran,which is itself bound to the DLC layer. Examples of pairs of classes ofmolecules, one of which may be immobilized as first molecule forinteraction with the other as second molecule are:

antigen/antibody

hormone/hormone receptor

polynucleotide strand/complementary strand

avidin/biotin

enzyme/substrate

carbohydrate/lectin

The sensor device may be used for the quantitative or qualitativedetermination of the second molecule in a sample applied to the sensingsurface, or may be used to study the interaction of the second moleculeswith the immobilized first molecules.

The invention will now be described in greater detail, by way ofillustration only, with reference to the accompanying drawings, in which

FIG. 1 is a schematic cross-sectional view of a first sensor deviceaccording to the invention;

FIG. 2 is a schematic view of chemical vapour deposition apparatus usedin the manufacture of the sensor device of FIG. 1; and

FIG. 3 is a schematic cross-sectional view of a second sensor deviceaccording to the invention.

Referring first to FIG. 1, a biosensor based on the principle offrustrated total reflection (FTR) comprises a substrate 1 in the form ofa glass chip, on the surface of which are formed successively a spacerlayer 2 of silicon oxide and a cavity layer 3 of silicon nitride. Thespacer layer 2 has a thickness of approximately 700 nm and the cavitylayer 3 a thickness of approximately 100 nm.

The surface of the cavity layer 3 is coated with a protective layer 4 ofdiamond-like carbon of approximate thickness 10 nm. Antibodies 5 arecovalently bound to the surface of the protective layer 4.

FIG. 2 shows apparatus used for the deposition of the spacer layer 2,cavity layer 3 and protective layer 4 on the substrate 1. The apparatuscomprises a vacuum chamber 10 with an exhaust port 11 formed in itsbase, the exhaust port 11 being connected to a pump 12. Gases are fed tothe chamber 10 through an inlet conduit 13, via a mass flow controller14. The inlet conduit 13 terminates in a “shower-head” arrangement 15which constitutes a first electrode.

A support 16 is positioned below the shower-head 15 and constitutes asecond electrode. The support is connected to a 13.65 MHz radiofrequencygenerator 18, via a matching unit 19.

In use, the substrate 1 is positioned on the support 16 and the sequenceof layers 2,3,4 built up sequentially by chemical vapour deposition. Agas is passed through the shower-head 15 into the space between theshower-head 15 and the support 16. A plasma is formed in that space anddeposition of ions created in the plasma takes place. First, the spacerlayer 2 is formed by introduction of an appropriate precursor gasthrough the shower-head 15 and appropriate setting of operatingparameters. The precursor gas and operating parameters are then changedto form the cavity layer 3. A further change of gas and operatingparameters leads to formation of the protective layer 4, as describedbelow.

In order to form the protective layer 4, methane gas is fed through theinlet conduit 13 as indicated by the arrow. The operating parameterswhich are used to control the extent and rate of deposition areprincipally the operating temperature, the flow rate of gas into thechamber 10, the pressure within the chamber 10 and the applied biasvoltage. A typical set of parameters is:

Temperature room temperature Flow rate 10 sccm (standard cubiccentimeters/minute) Pressure 50 mTorr Self-generated Bias voltage 70 V

With these operating conditions, deposition of a protective layer 4having a thickness of approximately 10 nm typically takes about 5minutes.

The arrangement described above, in which the workpiece (the substrate1) is placed on the driven electrode is unusual; a more conventionaldeposition arrangement being one in which the other electrode is driven.

Finally, FIG. 3 shows a second form of sensor device according to theinvention. This device is of the type described in WO 97/29362 andcomprises a substrate in the form of a chip 21 (eg of glass or silica)approximately 7 mm square and 2 mm in thickness. The chip 21 has arefractive index of 1.46. Coated on the upper surface of the chip 21 isa waveguide 22.

The interface between the chip 21 and the waveguide 22 is formed with aperiodic relief profile or grating 23 (the grating 23 is shown as beingsinusoidal though in practice it may be generally rectangular). Thewaveguide 22 is formed by deposition on the chip 21 and a correspondingrelief profile 24 may thus be formed also on the upper surface of thewaveguide 22. The upper surface of the waveguide 22 is coated with aprotective layer 26 of diamond-like carbon of approximate thickness 10nm (by a process similar to that described above in relation to theembodiment of FIG. 1). A layer 25 of biomolecules, eg antibodies, isimmobilized on the protective layer 26 in a known manner.

What is claimed is:
 1. A sensor device for detecting the binding offirst molecules to second molecules, said sensor device comprising: asensor surface, said sensor surface having an impervious layer ofdiamond-like carbon (DLC) applied thereto, and said impervious layerhaving first molecules immobilized thereon, wherein contact of a samplecontaining said second molecules with said sensor device results inbinding between said first molecules and said second molecules.
 2. Asensor device according to claim 1, wherein the DLC layer is formed by aplasma deposition or chemical vapour deposition technique.
 3. A sensordevice according to claim 2, wherein in said technique monomericstarting materials in the gas phase are introduced into a vacuum chambercontaining a pair of electrodes, a device to be coated being supportedin the chamber on one of the electrodes, and a radiofrequency ormicrowave discharge is applied.
 4. A sensor device as claimed in claim3, wherein the starting material includes a hydrocarbon, most preferablymethane.
 5. A sensor device as claimed in claim 1, wherein the DLC layerhas a surface which includes amino groups.
 6. A sensor device as claimedin claim 1, wherein the DLC layer has a surface which includescarboxylate groups.
 7. A sensor device as claimed in claim 1, whereinthe DLC layer has a thickness of less than 100 nm.
 8. A sensor device asclaimed in claim 1, wherein the DLC layer has a thickness of greaterthan 1 nm.
 9. A sensor device as claimed in claim 1, wherein the DLClayer has a thickness of the order of 10 nm.
 10. A sensor device asclaimed in claim 1, which is an optical sensor based on frustrated totalreflection, and comprising a) a cavity layer of transparent dielectricmaterial of refractive index n₃, b) a dielectric substrate of refractiveindex n₁, and c) interposed between the cavity layer and the substrate,a dielectric spacer layer of refractive index n₂.
 11. A sensor device asclaimed in claim 1, which is an optical sensor comprising a substratehaving a waveguide formed on at least part of the surface thereof, thewaveguide having a first major surface which constitutes an interfacebetween the waveguide and the substrate and a second major surface uponwhich the DLC layer is applied, at least a region of the first and/orsecond major surface being formed with a periodic refractive indexmodulation.
 12. A method for the analysis of biochemical species in afluid, which method comprises contacting a sample of the fluid withmolecules immobilized on the DLC layer of a sensor as claimed in claim1.
 13. A method of forming a sensor as claimed in claim 1, which methodcomprises supporting said sensor device on one of a pair of electrodeswithin a vacuum chamber, introducing monomeric starting material in thegas phase into the vacuum chamber and applying a radiofrequency ofmicrowave discharge between the electrodes.
 14. A sensor deviceaccording to claim 1, wherein said immobilized first molecules comprisebiomolecules.
 15. A sensor device according to claim 14, wherein saidbiomolecules comprise an antigen, hormone, polynucleotide strand,avidin, enzyme or carbohydrate.
 16. A sensor device according to claim14, wherein said second molecules comprise an antibody, hormonereceptor, complementary strand, biotin, substrate, or lectin.
 17. Asensor device according to claim 1, wherein said first molecules areimmobilized upon said layer of diamond-like carbon by being covalentlybound thereto.
 18. A sensor device according to claim 1, wherein saidlayer of diamond-like carbon contains amino groups and said firstmolecules are covalently bound to said amino groups.
 19. A sensor deviceaccording to claim 1, wherein said layer of diamond-like carbon containscarboxylate groups and said first molecules are covalently bound to saidcarboxylate groups.